Immobilized endoglycosidase fusion protein and use thereof

Abstract

A method for preparing an antibody-drug conjugate. The antibody-drug conjugate is formed by means of site-directed conjugation on the basis of an N-glycosylation site of an Fc region of an antibody. The method comprises: (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having a glycoside transfer activity; and (2) covalently linking the activated donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif by means of the catalytic action of the endoglycosidase; therefore, one-step conjugation is realized. The present invention also relates to an endoglycosidase fusion protein, which comprises a covalently linked endoglycosidase, a Halo tag, and/or a His tag. The present invention also relates to an immobilized endoglycosidase fusion protein obtained by means of immobilizing an endoglycosidase fusion protein on a support, a prepacked column filled with the immobilized fusion protein, and a method for purifying an antibody conjugate by using the immobilized endoglycosidase fusion protein.

The invention claimed is: 
     
       1. A method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is subjected to site-specific conjugation, based on an N-glycosylation site in an Fc region, and the method comprises the steps of:
 (1) providing a donor containing an oxazoline oligosaccharide, a protein containing the Fc region, and an immobilized endoglycosidase with glycosyltransferase activity; wherein the Fc contains a GlcNAc motif, wherein the GlcNAc motif is a glycan chain with N-acetylglucose-B-(1, 4)-N-acetylglucose; and 
 (2) covalently attaching the donor containing the oxazoline oligosaccharide to the protein containing the Fc region under the catalysis of the endoglycosidase, 
 wherein the donor containing the oxazoline oligosaccharide is a linker-payload compound of formula (I): 
 
       
         
           
           
               
               
           
         
         wherein P is a payload; 
         wherein D is the oxazoline oligosaccharide, wherein the oxazoline oligosaccharide has the following structure: first hexosyl derivative-(second hexosyl) f -β-D-glucopyranosyloxazoline, wherein carbon at position 6 of the first hexosyl derivative is in the form of—C(O)-; f is 0, 1, 2, 3, 4, 5 or 6; the β-D-glucopyranosyloxazoline has the following structure: 
       
       
         
           
           
               
               
           
         
         wherein L is a linker end, L is directly attached to carbonyl in D-C(O)-via-NH-therein, and the carbon at position 6 of the first hexosyl derivative in the form of—C(O)—is the same carbon in D-C(O) in formula (I), 
         wherein when L is an unbranched linker end, L is attached to one P, and t is 1; while when L is a branched linker end, each branch can be attached to one P, and t is an integer greater than 1, wherein the protein containing the Fc region is an antibody or an antigen-binding fragment thereof. 
       
     
     
       2. The method according to  claim 1 , wherein the protein containing the Fc region is an antibody. 
     
     
       3. The method according to  claim 1 , wherein the first hexosyl derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, and idosyl with carbon at position 6 of the first hexosyl in the form of —C(O)—; and/or
 the second hexosyl, at each occurrence, is independently selected from glucosyl, mannosyl, galactosyl, and fructosyl; and/or 
 individual monosaccharide moieties in an oligosaccharide structure are attached by β-(1→4) glycosidic bonds. 
 
     
     
       4. The method according to  claim 1 , wherein the oxazoline oligosaccharide has the following structure: first hexosyl derivative-B-D-glucopyranosyloxazoline, and wherein the first hexosyl derivative is mannosyl with carbon at position 6 in the form of —C(O)—; or
 the oxazoline oligosaccharide has the following structure: first hexosyl derivative-β-D-glucopyranosyloxazoline, and wherein the first hexosyl or its derivative is galactosyl with carbon at position 6 in the form of -C(O)-; or 
 the oxazoline oligosaccharide has the following structure: first hexosyl derivative-β-D-glucopyranosyloxazoline, and wherein the first hexosyl derivative is glucosyl with carbon at position 6 in the form of -C(O)-; or 
 the oxazoline oligosaccharide has the following structure: first hexosyl derivative-β-D-glucopyranosyloxazoline, and wherein the first hexosyl derivative is fructosyl with carbon at position 6 in the form of -C(O)-; or 
 the oxazoline oligosaccharide has the following structure: first hexosyl derivative-β-D-glucopyranosyloxazoline, and wherein the first hexosyl derivative is gulosyl with carbon at position 6 in the form of -C(O)-; or 
 the oxazoline oligosaccharide has the following structure: first hexosyl derivative-β-D-glucopyranosyloxazoline, and wherein the first hexosyl derivative is idosyl with carbon at position 6 in the form of -C(O)-. 
 
     
     
       5. The method according to  claim 1 , wherein the oxazoline oligosaccharide has the structure below: 
       
         
           
           
               
               
           
         
       
     
     
       6. The method according to  claim 1 
 wherein the antibody-drug conjugate has a structure as shown in formula (II): 
 
       
         
           
           
               
               
           
         
         R is hydrogen or α-L-fucosyl; 
         q is 1 or 2; and 
         Protein is the antibody or the antigen-binding fragment thereof. 
       
     
     
       7. The method according to  claim 6 ,
 wherein the antibody-drug conjugate has a structure as shown in formula (II-1), (II-2), (II-3), (II-4) or (II-5): 
 
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
         R is hydrogen or α-L-fucosyl; 
         q is 1 or 2; and 
         Ab is the antibody or the antigen-binding fragment thereof. 
       
     
     
       8. The method of  claim 7  wherein the antibody-drug conjugate has a structure as shown in formula (II-1): 
       
         
           
           
               
               
           
         
       
       and
 D-C(O)—is a disaccharide structure 
 
       
         
           
           
               
               
           
         
       
     
     
       9. The method according to  claim 1 , wherein-L-(P) t  is -L 2 -L 1 -B-P, that is, formula (I) is: 
       
         
           
           
               
               
           
         
         wherein 
         B is independently absent, or is 1) below, or 2) below, or a combination of 1) and 2) below: 1) A self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10  alkylene, C 4-10  cycloalkylene, C 4-10  heterocyclylene and—(CO)-; 
         L 1  is independently absent, or is an uncleavable sequence; or is a cleavable sequence comprising an amino acid sequence that is enzymatically cleavable, and the amino acid sequence that is enzymatically cleavable comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; 
         L 2  is independently absent; or is 1) below; or 2) below; or a combination of 1) and 2) below: 
         1) -NH-C 2-20  alkylene, wherein one or more -CH 2 - structures in the alkylene are optionally replaced by the following groups: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(═O) 2 -, -NR 5 -, -NR ⊕ R 6 R 7 -, C 4-10  cycloalkylene, C 4-10  heterocyclylene and phenylene, wherein the cycloalkylene, the heterocyclylene and the phenylene are each independently unsubstituted or substituted with at least one substituent selected from halogen, -C 1-10  alkyl, -C 1-10  haloalkyl, -C 1-10  alkylene-NH-R 8  and—C 1-10  alkylene-O-R 9 ; 
         2) an amino acid residue sequence, i.e., -*(AA) n **-, wherein n is an integer from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 to 100, AA, at each occurrence, is independently an amino acid residue, * represents an N-terminus of a corresponding amino acid, ** represents a C-terminus of the corresponding amino acid, and -(C 2 H 4 -O) m -(CH 2 ) p —is optionally present between amino and α-carbon of an amino acid, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and a * terminus forms an amide bond with the carbonyl in the disaccharide structure; 
         R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10  alkyl, and C 4-10  cycloalkylene; or R 1  and R 2  and carbon atoms attached thereto together form a 3 to 6-membered cycloalkylene, and/or R 3  and R 4  and carbon atoms attached thereto together form a 3 to 6-membered cycloalkylene; 
         P is a payload attached to moiety B, or moiety L 1 , or moiety L 2 . 
       
     
     
       10. The method according to  claim 1 , wherein -L-(P) t is 
       
         
           
           
               
               
           
         
       
       that is, formula (I) is: 
       
         
           
           
               
               
           
         
         wherein 
         Ld2 and each Ld1 are independently bonds, or are selected from —NH—C 1-20  alkylene-(CO)— and —NH-(PEG) i -(CO)—, or are natural amino acids independently unsubstituted or substituted with —CO—(PEG) j -R 11  on a side chain or oligomeric natural amino acids with a polymerization degree of 2 to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10); R 11  is C 1-10  alkyl; 
         d is 0, 1, 2, 3, 4, 5 or 6; 
         (PEG) i - and -(PEG) j - are each a PEG fragment, comprising a specified number of continuous —(O—C 2 H 4 )- structural units or continuous —(C 2 H 4 —O)— structural units, optionally with C 1-10  alkylene attached at one end; each i is independently an integer from 1 to 100, and each j is independently an integer from 1 to 100; 
         M is hydrogen or LKa-L 2 -L 1 -B-P; 
         Q is NH 2  or L 2 -L 1 -B-P; 
         provided that the following cases are excluded: M is hydrogen and Q is NH 2 ; 
         each LKa is independently selected from 
       
       
         
           
           
               
               
           
         
         opSu is 
       
       
         
           
           
               
               
           
         
       
       or a mixture thereof, wherein * represents a moiety attached to L 2 ;
 B is independently absent, or is 1) below, or 2) below, or a combination of 1) and 2) below: 1) A self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10  alkylene, C 4-10  cycloalkylene, C 4-10  heterocyclylene and—(CO)-; 
 L′ is independently absent, or is an uncleavable sequence; or is a cleavable sequence comprising an amino acid sequence that is enzymatically cleavable, and the amino acid sequence that is enzymatically cleavable comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; 
 L 2  is independently absent; or is 1) below; or 2) below; or a combination of 1) and 2) below: 
 1) -NH-C 2-20  alkylene, wherein one or more-CH 2 -structures in the alkylene are optionally replaced by the following groups:—CR 3 R 4 -, -O-, —(CO)-, -S-, -S(═O) 2 -, -NR 5 -, -N ⊕ R 6 R 7 -, C 4-10  cycloalkylene, C 4-10  heterocyclylene and phenylene, wherein the cycloalkylene, the heterocyclylene and the phenylene are each independently unsubstituted or substituted with at least one substituent selected from halogen, -C 1-10  alkyl, -C 1-10  haloalkyl, -C 1-10  alkylene-NH-R 8  and—C 1-10  alkylene -O-R 9 ; 
 2) an amino acid residue sequence, i.e., -*(AA) n **-, wherein n is an integer from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 to 100, AA, at each occurrence, is independently an amino acid residue, * represents an N-terminus of a corresponding amino acid, ** represents a C-terminus of the corresponding amino acid, and —(C 2 H 4 -O) m —(CH 2 ) p - is optionally present between amino and α-carbon of an amino acid, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and a * terminus forms an amide bond with the carbonyl in the disaccharide structure; 
 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10  alkyl, and C 4-10  cycloalkylene; or R 1  and R 2  and carbon atoms attached thereto together form a 3 to 6-membered cycloalkylene, and/or R 3  and R 4  and carbon atoms attached thereto together form a 3 to 6-membered cycloalkylene; 
 P is a payload attached to moiety B, or moiety L 1 , or moiety L 2 . 
 
     
     
       11. The method according to  claim 9 , wherein L 2  is the amino acid residue sequence, i.e., -*(AA) n **-, wherein n is an integer from 1 to 100, AA, at each occurrence, is independently an amino acid residue, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and —(C 2 H 4 -O) m —(CH 2 ) p —is optionally present between the amino and the α-carbon of the amino acid, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * terminus forms the amide bond with the carbonyl in the disaccharide structure. 
     
     
       12. The method according to  claim 1 , wherein the endoglycosidase with the glycosyltransferase activity is N-acetyl glucosamine endohydrolase, optionally wherein the N-acetyl glucosamine endohydrolase comprises at least one of Endo-S( Streptococcus pyogenes  endoglycosidase-S), Endo F3 ( Elizabethkingia miricola  endoglycosidase-F3), Endo S2 (Endoglycosidase-S2,  S. pyogenes  endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd,  S. pyogenes  endoglycosidase-Sd) and Endo CC (Endoglycosidase-CC,  S. pyogenes  endonuclease-CC); or
 the N-acetyl glucosamine endohydrolase comprises at least one of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S and Endo S2. 
 
     
     
       13. The method according to  claim 1 , wherein the endoglycosidase with the glycosyltransferase activity is covalently attached to a Halo tag, and is immobilized on a support containing haloalkyl linker via the Halo tag, and the Halo tag is a dehalogenase or its variant or truncated functional active moiety, optionally wherein one end of the endoglycosidase is covalently attached to the Halo tag, and the other end is covalently attached to a His tag; or
 an amino end of the endoglycosidase is covalently attached to the Halo tag, and a carboxyl end is covalently attached to the His tag, i.e., Halo-endoglycosidase-His; or 
 the amino end of the endoglycosidase is attached to the Halo tag, the carboxyl end is attached to the His tag, and the endoglycosidase is Endo-S2, i.e., Halo-Endo S2-His. 
 
     
     
       14. The method according to  claim 13 , wherein the support comprises a chloroalkyl linker, such that the endoglycosidase is immobilized on the support under the covalent interaction between the chloroalkyl linker and the Halo tag, optionally wherein the chloroalkyl linker is generated by a chloroalkyl substrate with a structure of formula (III): 
       
         
           
           
               
               
           
         
         wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19. 
       
     
     
       15. The method according to  claim 13 , wherein the support has a structure of formula (IV): 
       
         
           
           
               
               
           
         
         wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19; 
            is resin, a bead, a membrane, gel, a matrix, a film, a plate, a well, a tube, a glass slide or a surface. 
       
     
     
       16. The method according to  claim 15 , wherein the resin is selected from agarose resin, silicone resin, polymethyl methacrylate resin and cellulose resin. 
     
     
       17. The method according to  claim 15 , wherein the resin is highly cross-linked agarose resin or polymethyl methacrylate resin.